Summary
Conventionally, expression plasmids in Escherichia coli have generally been constructed using ligation reaction-assisted cloning followed by the generation of inserts. In such cases, the insert was generated by polymerase chain reaction (PCR), digestion using restriction enzymes, or oligonucleotide synthesis. To overcome the restrictions of these conventional methods, we improved them by utilizing an in vitro site-specific recombination reaction, based on the integrase–excisionase system of bacteriophage λ to insert DNA fragments. This method enabled us to insert tens of fragments into expression vectors in parallel. We applied these methods to produce glutathione S-transferase (GST)-fused or maltose-binding protein (MBP)-fused proteins in Escherichia coli. As a result, we successfully produced and purified more than 3,000 recombinant proteins for further study of reverse chemical genetics.
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Acknowledgments
We are grateful to Dr. Takahiro Nagase for his truly helpful discussions, invaluable suggestions, and encouragement. The author also thanks Miss Tomomi Tajino and Mr. Kazuhiro Sato for their technical assistance. This study was supported by grants from the CREATE Program (Collaboration of Regional Entities for the Advancement of Technological Excellence) from JST (Japan Science and Technology Corporation); by the Genome Network Project of the Ministry of Education, Culture, Sports, Science, and Technology of Japan; and by the Kazusa DNA Research Institute.
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Shimada, K., Koga, H. (2009). High-Throughput Production of the Recombinant Proteins Expressed in Escherichia coli Utilizing cDNA Resources. In: Koga, H. (eds) Reverse Chemical Genetics. Methods in Molecular Biology™, vol 577. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-232-2_7
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DOI: https://doi.org/10.1007/978-1-60761-232-2_7
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